Analysis of the Upregulated Expression Mechanism of Apoptotic Chromatin Condensation Inducer 1 in Hepatocellular Carcinoma Based on Bioinformatics

Background/Aims: A large number of differentially expressed molecules exist in hepatocellular carcinoma (HCC), and the mechanism by which they upregulate or downregulate expression is still unclear. The purpose of this study is to explore the possible mechanism of differential expression of apoptotic chromatin condensation inducer 1 (Acin1) in HCC. Materials and Methods: A mouse HCC model was constructed, and the expression of Acin1 in HCC was analyzed by whole transcriptome sequencing, bioinformatics analysis, and reverse transcription-quantitative polymerase chain reaction, and differentially expressed Acin1-related genes were screened to construct a protein–protein interaction and competing endogenous RNA (ceRNA) network. The microRNA (miRNAs) targeting Acin1 were further predicted using online databases and finally compared with sequencing data. Results: The expression of Acin1 was significantly up-regulated in HCC compared to the paracancerous and healthy control groups (P < .001). The top 10 upregulated genes closely related to Acin1 (Slc3a2, Wiz, Srrm2, Akt1, Hnrnpu, Sap18b, Pabpn1, Ddx39b, Eif4a3, and Rnps1) were mainly involved in pathways such as messenger RNA (mRNA) surveillance, RNA transport, spliceosome, Janus kinase/signal transducers and activators of transcription signaling, apoptosis, and ubiquitin-mediated proteolysis. The ceRNA network identified several molecules (2 long noncoding RNAs, 50 miRNAs, and 49 mRNAs) interacting with Acin1, among which miR-674-5p was highly expressed in all sample tissues, and higher than that of other differentially expressed miRNAs, and significantly downregulated in HCC. Multiple online databases such as miRWalk also predicted that miR-674-5p targets Acin1. This shows that miR-674-5p may be an important molecule for targeting Acin1. Conclusion: Acin1 is overexpressed in HCC, and the overexpressed Acin1 is most likely regulated by miR-674-5p and other ceRNA molecules.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common and deadly malignant tumors in the world, ranking third in terms of mortality among all cancers. 1 It poses a significant threat to human health.At present, with the development of multi-omics research technology, a large number of differentially expressed molecules have been found in HCC tumor tissues and surrounding tissues.3][4] However, the mechanisms by which these differentially expressed molecules are up-or downregulated are not well understood.In recent years, relevant factors involved in messenger RNA (mRNA) stability, splicing, and translation have been proved to play an important role in the occurrence and development of tumors. 5Apoptotic chromatin condensation inducer 1 (Acin1) is an RNA-binding protein, also known as Acinus, which was initially found to induce caspase-3 activation to promote apoptotic chromatin condensation and densification, thereby participating in nuclear fragmentation during apoptosis. 6,7It was later found that this protein was also a component of the splicing-dependent multiprotein exon junction complex (EJC) deposited on spliced mRNAs and was involved in splicing-related mRNA metabolism.It plays an important role in posttranscriptional regulation of genes and the expression of specific genes by downregulating nuclear mRNA splicing through the spliceosome, where the spliceosome machinery stops and prevents or reduces mRNA splicing. 8,9Recent studies have revealed that Acin1 plays a role in the physiology and pathology of a variety of diseases and cancers.
Lin et al 6 found by whole transcriptome analysis that the expression of Acin1 transcript decreased and the splicing patterns changed during the development of brown adipose tissue.This change of Acin1 transcript differentially regulated the formation of brown adipose tissue in fibroblasts.In 2018, Xue et al 8 found that the expression level of Acin1 mRNA in platelets of lung cancer patients was significantly higher than that of healthy control group, and the change in its level had potential clinical value for the diagnosis of lung cancer.In addition, the fusion of the Acin1-Nutm1 gene may also cause cryptic chromosomal rearrangements, which play an important role in infant acute lymphoblastic leukemia. 10It is evident that Acin1 has an increasingly obvious role in cancer.
However, the involvement of Acin1 in HCC has not been reported yet, so this study analyzed the expression of Acin1 mRNA in HCC by whole transcriptome sequencing, bioinformatics analysis, and reverse trans cript ion-q uanti tativ e polymerase chain reaction (RT-qPCR), further screened the differentially expressed Acin1-related genes in tumor tissues, constructed protein-protein interaction (PPI) and competitive endogenous RNA (ceRNA) regulatory networks, etc., and further analyzed their functions and interactions through online databases to predict the targeting and regulation of Acin1.We will further screen the differentially expressed Acin1-related genes in tumor tissues, construct PPI and competing endogenous RNA (ceRNA) regulatory networks, analyze their functions and interactions, and further predict the miRNAs targeting Acin1 through online databases to elucidate the possible molecular mechanisms of Acin1 gene aberrant expression in HCC.

MATERIALS AND METHODS Construction of Hepatocellular Carcinoma Mouse Model, Samples Sequencing, and Data Analysis
One hundred healthy mice aged 6-8 weeks and weighing 25-30 g were used to construct the mouse model of HCC according to the previous method in our laboratory. 11he tumor tissues (set up as group "H") and paracancerous liver tissues (set up as group "C") were collected separately, as well as liver tissues from control mice injected with saline (set up as group "OO").Three biological replicates of 100 mg of each sample were taken, and total RNA was extracted using a RNA extraction kit and sent to BGI Genomics Co., Ltd.(hereinafter referred to as "Huada Gene").The sequencing data were analyzed online using Dr. Tom multi-omics data mining platform (https ://bi osys.bgi.c om/).This study was approved by the Ethical Committee of Youjiang Medical University for Nationalities (Ethics Review No. 20201205026, Date: 2020-12-05).

Reverse Trans cript ion-Q uanti tativ e Polymerase Chain Reaction to Detect the Relative Expression of Apoptotic Chromatin Condensation Inducer 1
Total RNA from each sample tissue was reverse transcribed to cDNA and then subjected to real-time fluorescence PCR.The qPCR procedure included predenaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing/extension at 60°C for 30 seconds, for a total of 40 cycles.The lysis curve reaction program was set up according to the recommended program for the Roche LightCycler96 RT-qPCR instrument, and relative expression values were calculated using 2 -ΔΔCt .The expression histograms were plotted using GraphPad Prism version 8.0.The internal reference of RT-qPCR was Gapdh.The primer sequences of Acin1 and Gapdh are as follows: the forward primer of Acin1 was 5"-GACTCTTCCGTAAGACTTAA GG-3: " and the reverse primer was 5"-TTCTCTTCGTTCTTCGC-3"; the forward primer of Gapdh was 5"-GTTGTCTCCTGCGACTCA-3" and the reverse primer was 5"-TGGTCCAGGTTCTTACTC-3".

Apoptotic Chromatin Condensation Inducer 1-Related Gene Screening
Differentially expressed Acin1-related genes were screened by the Dr. Tom multi-omics data mining platform to construct PPI networks and ceRNA regulatory

Main Points
• Compared with the paracancerous group and healthy control group, the expression of apoptotic chromatin condensation inducer 1 (Acin1) was significantly upregulated in hepatocellular carcinoma (HCC) (P < .001).

•
The ceRNA network analysis revealed that Acin1 interacted with several molecules as competing endogenous RNAs (ceRNAs), including 2 long noncoding RNAs, 50 microR-NAs (miRNAs), and 49 mRNAs.Among them, miR-674-5p was highly expressed in all sampled tissues, surpassing the expression of other differentially expressed miRNAs, and significantly downregulated in HCC.Various online databases, such as miRWalk, also predicted the targeting of Acin1 by miR-674-5p.This suggests that miR-674-5p may be an important molecule for targeting Acin1.
networks.The screening thresholds for differentially expressed genes were: |log2 (FPKM ratio)| > 1, Q < .05(Q is the corrected P value and FPKM, fragments per kilobase of transcript per million fragments mapped).The FPKM refers to reads per kilobase of exon model per million mapped reads, that is, the number of reads per 1000 exons per 1 million reads on the pair.The number of reads per kilobase of exon model per million mapped reads.

Functional Enrichment of Apoptotic Chromatin Condensation Inducer 1-Related Genes and Protein Interaction Network Map Construction
Using Dr. Tom multi-omics data mining platform, we constructed a heat map of the expression of Acin1 and its related genes in tumor tissues, paracancerous tissues, and healthy mouse liver tissues, analyzed the ceRNA regulatory network and PPI network of Acin1 and its related genes, and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on Acin1 and its related genes.The PPI network maps were further visualized using the String online database (http: //str ing.e mbl.d e/).

Prediction of microRNAs Targeting the Regulation of Apoptotic chromatin condensation inducer 1
MiRWalk (http: //mir walk.umm.u ni-he idelb erg.d e/), Starbase (https ://st arbas e.sys u.edu .cn/), miRecords (http: //mir ecord s.bio lead.org/) , and TargetScan (http: //www .targetsca n.org /) were used to predict miRNAs targeting Acin1, and the predicted results were intersected using the jvenn online tool (http: //jve nn.to ulous e.inr a.fr/ app/e xampl e.htm l) to screen for possible miRNAs.Prediction conditions: Acin1 as target gene, selected mouse species, other conditions set by default.The results obtained were further analyzed in combination with the expression of miRNAs in the sequencing data.

Statistical Analysis
Statistical Package for the Social Sciences Statistics version 23.0 statistical software (IBM Corp., Armonk, NY, USA) was used to analyze the measurement data.The measurement data were expressed as mean ± SD.After the normal distribution and variance homogeneity test, the data meeting the normal distribution and variance homogeneity were tested by 2 independent samples t-tests, and the difference was statistically significant with P < .05.

Expression of Apoptotic Chromatin Condensation Inducer 1 Gene
The results of sequencing data showed that Acin1 was expressed in all sample tissues, and Acin1 mRNA was significantly upregulated in tumor tissues compared with paracancerous tissues and healthy control tissues (P < .001).The expression of Acin1 mRNA was further verified by an RT-qPCR experiment.The results showed that Acin1 mRNA was also significantly upregulated in tumor tissue (P < .001)(Figure 1).

Interaction Networks and Expression of Apoptotic Chromatin Condensation Inducer 1-related Genes
The results of interaction analysis of Acin1 and its related genes showed that there were a large number of proteins/ genes with PPI relationship with Acin1, including 37 closely related proteins/genes (Figure 2).The clustered heat map showed that among the 37 genes, Slc3a2, Wiz, Srrm2, Akt1, Hnrnpu, Sap18b, Pabpn1, Ddx39b, Eif4a3, Rnps1, Api5, Zc3h18, and Srrm1 were significantly upregulated in tumor tissues compared with those in the paracancerous tissues and healthy control tissues.The intergroup clustered heat map (Figure 3A) and inter-sample clustered heat map (Figure 3B) of the expression of Acin1 and its related genes were generally consistent (Figure 3), and the top 10 Acin1-related genes expressed in HCC are Slc3a2, Wiz, Srrm2, Akt1, Hnrnpu, Sap18b, Pabpn1, Ddx39b, Eif4a3, and Rnps1.It can be seen that these 10 genes may play an important role in the regulation of the occurrence and development of HCC by Acin1.

Functions and Pathways Involved in Apoptotic Chromatin Condensation Inducer 1 and its Related Genes
The functional enrichment analysis of Acin1 and its related genes found that they mainly play a role in the nuclear speck, nucleus, nucleoplasm, spliceosomal complex, EJC, and cytoplasm, etc., participate in biological processes such as mRNA processing, RNA splicing, spliceosomal complex assembly and apoptosis regulation, etc., and play a role in small ubiquitin-related modifier (SUMO) transferase activity, SUMO ligase activity, poly(A) binding, nucleic acid binding, and nucleotide binding.The results of pathway enrichment showed that they were mainly involved in mRNA surveillance, RNA transport, spliceosome, Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway, apoptosis, and ubiquitinmediated proteolysis (Figure 4).

MicroRNAs and Competing Endogenous RNA Network Targeting and Regulating Apoptotic Chromatin Condensation Inducer 1
This study further analyzed the miRNAs that target to regulate Acin1 and ceRNAs that can competitively combine with miRNA and mRNA to regulate Acin1.Through the analysis of the ceRNA regulatory network, we found 101 ceRNAs molecules related to Acin1, including 2 long noncoding RNAs (4930533N22Rik and Gm21269), 50 miRNAs, and 49 mRNAs (Table 1).Many molecules have targeted regulatory relationships (Figure 5).Among many miRNAs targeted to regulate Acin1, we found 3 miRNAs that were significantly negatively correlated with the expression of Acin1, namely miR-6395, miR-674-5p, and miR-7067-5p, of which miR-674-5p was expressed at a higher level in all tissue samples, and its expression was much higher than other differentially expressed miRNAs and was significantly downregulated in tumor tissues relative to paracancerous and healthy control tissues.
Further, we used 4 online databases, miRWalk, StarBase, miRecords, and TargetScan, to comprehensively predict the miRNAs targeting Acin1, and found that all 4 databases also predicted miR-674-5p targeting Acin1 (Figure 6).Therefore, we speculate that miR-674-5p may be an important regulator of Acin1 overexpression, and that multiple ceRNA molecules in the ceRNA regulatory network that are associated with Acin1 may also play an important role in its overexpression.These molecules may work coordinately to finely regulate Acin1 expression.

DISCUSSION
Genetic and acquired changes in selective splicing of mRNA precursors play an important role in the development of human disease and many cancer-related genes are regulated by alternative splicing. 12,13Understanding abnormal splicing or splicing variation will help us understand the causes of malignant transformation.Apoptotic chromatin condensation inducer 1 is a multifunctional nuclear protein, an auxiliary component of the splicingdependent multiprotein EJC, deposited at the splice junctions of mRNAs, which confers the binding of RNA to the complex and has an important role in apoptosis, selective RNA splicing, and basal autophagy. 14It has been shown that the molecule is widely present in a variety of tissues and organs, including glandular vesicles, the retina, blood, lungs, liver, [15][16][17][18][19] etc.Our results also found that Acin1 was expressed in liver tissue and was significantly overexpressed in HCC tumor tissue.We know that splice errors that lead to the production of abnormal transcripts rarely occur in normal cells, but research has found that they are almost the inherent characteristics of cancer cells. 20Tumor-specific splicing alterations are created by mutations that disrupt splicing-regulatory elements within genes and impair splicing recognition or by altering the RNA-binding preferences of individual splicing factors. 21Apoptotic chromatin condensation inducer 1 has the function of participating in splicing-related mRNA metabolism, but it is rarely reported that Acin1 participates in HCC.In this article, we explore the causes and possible molecular mechanisms of the overexpression of Acin1 in HCC in order to provide some insights into the pathogenesis of HCC.
6][27] In order to explore the cause of the overexpression of Acin1 in HCC, we further analyzed the miRNAs that target to regulate Acin1 and ceRNAs that can competitively combine with miRNA and mRNA to regulate Acin1.The results showed that many miRNAs can target and regulate Acin1.Among them, we found that the expression of miR-674-5p in all sample tissues was higher, and its expression was much higher than other differentially expressed miRNAs and was significantly downregulated in tumor tissues relative to paracancerous and healthy control tissues.This is negatively correlated with the expression of Acin1.Further, we also predicted that miR-674-5p is the miRNA that targets and regulates Acin1 by using several classic miRNA online prediction databases such as miRWalk, StarBas, miRecords, and TargetScan.Therefore, we speculated that miR-674-5p may play a crucial role in the pathogenesis of HCC by targeting Acin1.However, further experiments are warranted to confirm the regulatory relationship between miR-674-5p and Acin1 in HCC development.It has been reported that miR-674-5p/5-LO axis can be involved in the autoimmune response to acute liver injury in mice induced by cutanocin A (cona).This study indicated that the expression of miR-674-5p was significantly downregulated in the liver damaged by cona. 28In our study, miR-674-5p is downregulated in HCC, while Acin1 is upregulated in HCC, which shows that miR-674-5p is likely to regulate the upregulation of Acin1.However, the functional role of genes is also often influenced by multiple interacting molecules.In order to further study the possible molecular mechanism of Acin1 participating in HCC, we also investigated the effect of ceRNA.The ceRNA regulatory network plays an important role in HCC. 29In our study, multiple molecules were found to interact with Acin1 as ceRNAs, including 2 lncRNAs (4930533N22Rik and Gm21269), 50 miR-NAs, and 49 mRNAs.These findings suggest that the regulatory mechanism of ceRNA may be significant in the upregulation of Acin1 and possibly contribute to its finetuned regulation.
In summary, this study revealed that Acin1 is overexpressed in HCC through the utilization of a mouse model of HCC, whole transcriptome sequencing, bioinformatics analysis, and RT-qPCR experiments.It was found that the upregulation of Acin1 may be regulated by miR-674-5p and multiple ceRNA molecules.They may contribute to the development of HCC by exerting molecular functions such as protein binding, nucleic acid binding, and ubiquitination-like modifications, affecting pathways such as mRNA surveillance, RNA transport, spliceosome, JAK-STAT signaling pathway, apoptosis, and ubiquitinmediated proteolysis.However, the specific mechanisms through which miR-674-5p and these ceRNA molecules regulate the involvement of Acin1 in HCC necessitate further investigation in order to obtain a comprehensive understanding of their roles in HCC development.

Figure 2 .
Figure 2. The protein-protein interaction network of apoptotic chromatin condensation inducer 1 and its related genes.

Figure 3 .
Figure 3. Inter-group clustered heat map (A) and inter-sample clustered heat map (B) of apoptotic chromatin condensation inducer 1 and its related genes.Note: The expression in this figure refers to the fragments per kilobase of transcript per million fragments mapped value.

Figure 4 .
Figure 4. Enrichment analysis of GO and KEGG of Acin1 and its related genes.Note: A: biological process; B: cellular component; C: molecular function; D: KEGG pathway.Acin 1, apoptotic chromatin condensation inducer 1; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5 .
Figure 5.The competing endogenous RNA network of apoptotic chromatin condensation inducer 1 gene and its related genes in hepatocellular carcinoma.lncRNA, long noncoding RNA; mRNA, messenger RNA; miRNA, microRNA.

Table 1 .
The Competing Endogenous RNA Molecules Related to Apoptotic Chromatin Condensation Inducer 1